The field of hematology involves the study of blood, including the discrete cell type populations that make up the blood. One important tool in this field involves the ability to reliably and accurately distinguish and count the various cell type populations. Clinical significance can be attributed to abnormal levels, in both relative and absolute terms, of most, if not all cell type populations.
In particular, the study of the kinetics of blood cell production and destruction depends on an assessment of the number of new cells being delivered to the circulation per unit time. New red cells (known as reticulocytes) are easily identified and can be quantified as a percentage of total red blood cells ("RBC's"). This percentage can be multiplied by the red cell count in order to provide the number per microliter.
In order to identify reticulocytes, the cells can be distinguished from mature red blood cells by the presence in reticulocytes of a distinctive stainable marker formed of mitochondria, ribosomes, and other cytoplasmic organelles. This marker is visible when precipitated by so-called supravital dyes, such as new methylene blue, brilliant cresyl blue, and acridine orange.
Reticulocytes require on the order of 3 to 4 days for maturation into mature (non-stainable) red blood cells, with about 2 to 3 days of this period being spent in the marrow itself, and about one day in the peripheral blood. Since approximately 1% of the circulating red cells in normal humans are replaced daily, and a newly released erythrocyte is identifiable as a reticulocyte for only about one day, it follows that under normal circumstances about 1% of circulating red cells are detectable as reticulocytes.
Variations in the reticulocyte count can be clinically significant, if detected with assurance. Levels approaching 3% (based on the total number of circulating red cells) are seen in instances of increased marrow activity, e.g., when blood synthesis is stimulated as by erythropoietin. In contrast, levels below about 0.5% can be an indication of bone marrow incompetence. Hence, a reticulocyte count is an effective measure of marrow erythroid output, since the release of one reticulocyte signals the production of one red cell by the marrow.
A wide variety of approaches have been described for determining absolute and/or relative reticulocyte levels. Although such approaches have traditionally been performed manually, automated procedures are being used with increasing frequency. An increasing number of major instrument manufacturers of automated cell counting instruments have added the ability to count reticulocytes to their systems in recent years. Some instruments provide values for what is known as a "reticulocyte maturation index", or "RMI". Although this parameter is not yet standardized for clinical reporting, it nevertheless shows great promise as a clinically useful parameter.
A common principle underlying the use of each method is the need for a reliable, standardized reticulocyte control composition, against which to assess the reliability of the results. Presently, reticulocyte controls are provided in a number of different forms. Human blood itself is generally considered to be unsuitable for the preparation of reticulocyte controls. Reasons for this include the safety concerns associated in recent years with the use of human blood products, as well as the fact that human blood is typically too low in reticulocyte count to be useful for the preparation of a wide range of control levels.
A control composition known as "Retic-C".TM. is available in control levels I, II and III from Coulter Corporation (Miami, Fla.). This product includes avian red blood cells as the retigulocyte analogues. Such cells are not derived from true reticulocytes, however, nor do they stain in the manner common to reticulocytes. As a result, the composition is limited in use to automated instruments that rely on detection techniques other than traditional staining, such as the Coulter "STK-S".TM. instrument. The avian cells are significantly larger than human reticulocytes and, in contrast to reticulocytes, also contain a nucleus.
Another control composition is a reticulocyte analogue product available as the "Retic-CHEX".TM. product in concentrations of 1, 3, and 5% from Streck Laboratories (Omaha, Neb.). This control also suffers from a number of drawbacks, however, including poor staining intensity, and performance that is only minimally like that of true reticulocytes. Yet other control compositions, both of which are manufactured by Streck, include "Test Point".TM. product (available in 2 and 5% levels for use with Miles/Technicon instruments), and a 2 and 5% level control available for instruments available from Sysmex. Both compositions are limited in that they provide only two levels, with the upper level being significantly lower than may be desired.
As a result, there do not appear to be any control compositions available on the market that provide an optimal combination of stability, true reticulocyte appearance and stain characteristics, and wide utility with most, if not all, presently available assay techniques. A reticulocyte control product capable of providing such attributes would be highly desirable.